Transcriptomic profiling and gene network analysis revealed regulatory mechanisms of bract development in Bougainvillea glabra

Background Bracts are important for ornamental plants, and their developmental regulation process is complex; however, relatively little research has been conducted on bracts. In this study, physiological, biochemical and morphological changes in Bougainvillea glabra leaves, leaf buds and bracts during seven developmental periods were systematically investigated. Moreover, transcriptomic data of B. glabra bracts were obtained using PacBio and Illumina sequencing technologies, and key genes regulating their development were screened. Results Scanning electron microscopy revealed that the bracts develop via a process involving regression of hairs and a color change from green to white. Transcriptome sequencing revealed 79,130,973 bp of transcript sequences and 45,788 transcripts. Differential gene expression analysis revealed 50 expression patterns across seven developmental periods, with significant variability in transcription factors such as BgAP1, BgFULL, BgCMB1, BgSPL16, BgSPL8, BgDEFA, BgEIL1, and BgBH305. KEGG and GO analyses of growth and development showed the involvement of chlorophyll metabolism and hormone-related metabolic pathways. The chlorophyll metabolism genes included BgPORA, BgSGR, BgPPH, BgPAO and BgRCCR. The growth hormone and abscisic acid signaling pathways involved 44 and 23 homologous genes, and coexpression network analyses revealed that the screened genes BgAPRR5 and BgEXLA1 are involved in the regulation of bract development. Conclusions These findings improve the understanding of the molecular mechanism of plant bract development and provide important guidance for the molecular regulation and genetic improvement of the growth and development of ornamental plants, mainly ornamental bracts. Supplementary Information The online version contains supplementary material available at 10.1186/s12870-024-05246-7.


Introduction
Bracts are metamorphosed leaves that accompany the inflorescence.In general, any leaf associated with an inflorescence can be defined as a bract [1,2].Strictly speaking, the bract is not an inflorescence structure; rather, it can be considered an extension of floral organs.The bract primordia originating from the stem meristem are usually generated at the very early stage of reproductive development [3].However, a "bract suppression system" has been observed in many, but not all, angiosperm species.This system stops the development of the bract and eventually subsumes the bract primordia into the floral meristem.Therefore, bracts are absent in many model plants, such as Arabidopsis, maize (Zea mays) and rice (Oryza sativa).The bract development system has been suppressed rather than removed in these taller plants.In rice spikes, SPLs and NL1 synergistically regulate the transcriptome and chromatin accessibility after the reproductive transition, as well as the expression of other bract repressor genes, such as PLA1, PLA2, and RI.PLA2 and PLA3 are integrated into common downstream X factors (one or more unknown genes) to regulate bract repression.Bract repression is not only necessary for flower development but also essential for the transition from asexual to reproductive meristems [4].A study in rice indicated that bract suppression is not only necessary for floral development but also critical for the transition from vegetative to reproductive branching [5].Although bract suppression is a conserved mechanism in most angiosperm lineages, some plants, such as species belonging to the Cornaceae, Nyssaceae, Nyctaginaceae and Araceae families, exhibit natural bract development.
In many ornamental plants, the bract is an important organ that participates in reproductive events, as well as an ornamental feature [6].Bracts with ornamental value are called petaloid bracts, formed as a result of the ectopic expression of genes determining petal identity in the leaf.This process is thought to involve developmental signal transduction and the activation or suppression of related genes.Ectopic petalization (the development of petal-like characteristics in nonpetal organs) contributes to the diversity of flower morphology in the process of angiosperm evolution.Bract development appears to occur due to regulation by ABC/ABCDE class genes of the MADS-box family.Sepal formation is determined by the AGAMOUS-like 6 (AGL6) and Septin (SEP) genes, but few specific studies have been conducted on this subject.The MADS-box gene family belonging to the B-class of the ABC model has been verified to play a key role in ectopic petalization.A MADS-box gene, AGL6, was shown to regulate floral organ development and flowering time in Arabidopsis [7].Overexpression of the AGL6 gene in Arabidopsis promoted the growth of petal-like bracts.Similarly, ectopic expression of MADS-box genes was observed in Cornus officinalis.Significant upregulation of the CorPI-B, CorPI-A and CorAP3 genes was detected in C. officinalis bracts during different developmental stages [8,9].In Aristolochia (Aristolochiaceae, basal angiosperm), the expression of an AP3-like gene was detected only during the late stages of petaloid perianth development, and the expression of B-class paralogs was detected in the late stage of Aquilegia petaloid sepal development [10].The JAGGED (JAG) gene is the only gene reported to positively regulate bract development in Arabidopsis.Overexpression of the JAG gene promoted bract development in Arabidopsis, and bract development was inhibited in the APETALA1 (AP1) and JAG double mutant [11].A recent study identified a bract-specific gene, DiASR1 (abscisic acid-, stress-and ripening-related protein), from the dove tree (Davidia involucrata).Overexpression of the DiASR1 gene induced the formation of bract-like leaves in Arabidopsis [2].Although there is a large body of literature on bract development, most of the research focused on bract inhibitory systems and was carried out in model plants, whereas the mechanism of natural bract development remains largely unknown.More genes regulating bract development are expected to be explored in naturally bracted species.
Ornamental plant bracts are currently being studied mainly for their pigments.For example, Bougainvillea, a woody plant of Nyctaginaceae [12] that is rich in pigments such as chalcone-flavonone isomerase 1 (CHI1), 4,5-DOPA dioxygenase extradiol (DOD) and flavanone 3-hydroxylase (F3H), has been shown to be involved in bract color change [13,14].However, there is very little research on how bracts develop, and the molecular mechanism underlying the key events of bract development, including organogenesis, chloroplast degeneration, petal identity determination, rapid growth and abscission, needs to be holistically investigated.Bougainvillea glabra 'Mrs.Eva White' bracts have significant ornamental value.They are large and thin, making them ideal for studying bract development.To elucidate the developmental mechanisms of bracts in ornamental plants, we conducted a comprehensive analysis of B. glabra 'Mrs.Eva White' at the morphological and transcriptomic levels to identify key genes involved in these processes.

Morphological and photosynthetic pigment changes during bract development in B. glabra
The development of B. glabra buds occurs in five main stages, as observed by resin sectioning.At the initial stage, only bracts and growth points are present (Fig. S1A-a).The primordium forms the outermost whorl of the inflorescence, around which the primordium of the bracts appears in rapid succession and consists of outer and inner cells, with the former being larger and looser and the latter being smaller and more compact, followed by gradual growth of the flower primordium (Fig. 1B-a).The floret primordium rapidly differentiates (Fig. S1A-b), and the perianth whorl begins to appear around the primordium (Fig. 1A, S1A-b).As the floral organs develop further, the buds expand, the sepals and petals gradually elongate, the growth cone broadens, the outer petals differentiate into small projections, and the stamen primordia begin to form (Fig. 1B-c).During the LB (late bud) period, the stage of bract protocorm differentiation, the androecium has not yet completed its differentiation and is still in the protocorm stage (Fig. S1B-a).Subsequently, androgynous differentiation is completed, and this stage is known as the stage of floral primordium differentiation (Fig. S1B-b).
Scanning electron microscopy revealed that bract villi gradually degenerated during development (Fig. 1C).During the BR1 period, the villus density was high, and the density gradually decreased as the bracts developed (Fig. S1C).The development of B. glabra bracts showed a slow-fast-slow pattern.The bracts were initially green and then showed a change in chlorophyll degradation.From the onset of bract primordium formation, growth was slow for the first 5 days, accelerating on day 6 and entering a period of rapid growth on day 17, when the bracts gradually changed from green to white (Fig. 1D).The chlorophyll content tended to decrease during development, especially significantly after bract period 2, indicating that bract development was accompanied by a process of chlorophyll regreening.However, the chlorophyll content of the bracts increased slightly during senescence.Overall, the chlorophyll and carotene contents of the B. glabra bracts were significantly lower than those of the leaf blades (Fig. 1E).

Transcriptome sequencing and sequence analysis of leaf buds, leaves and bracts at different developmental stages
In this study, the samples were subjected to transcriptome sequencing using the circular consensus sequence technique, which yielded a circular consensus sequence with a total length of 1041,643,869 bp.The homologs and polyA tails were removed by IsoSeq processing, and 463,177 FLNC reads were obtained.Subsequently, the FLNC sequences were clustered and demultiplexed using the ICE tool of SMRTlink software, resulting in a nonredundant transcript sequence of 79,134,466 bp.Thereafter, to improve sequence accuracy, the transcript sequence was further corrected using LoRDEC error correction software, resulting in a corrected transcript sequence of 79,130,973 bp.
After obtaining the transcript sequences, they were clustered and demultiplexed using cd-hit software to create the final full-length transcript sequences, which were used as the reference transcript sequences for second-generation data comparison.A total of 45,788 transcripts were detected in the samples, with a total of 706,647,796 bp and an average length of 1,544 bp.These findings provide an important database for subsequent transcriptome analyses.

Transcriptomic differential gene expression analysis
Principal component analysis (PCA) of the gene expression levels (FPKM) of all the samples revealed that the samples from the LB, FB and BR1 periods clustered together, whereas those from the BR2 period began to show a dispersion trend.Over time, samples from the BR3, BR4 and BR5 periods showed more similar gene expression patterns.However, samples from the LE and BR6 periods showed a clear trend of separation from the other periods (Fig. 2A).
Analyses of differentially expressed genes between LB and FB, FB and BR2, BR2 and BR5 and BR5 and LE revealed 245 differentially expressed genes between LB and FB and 2,039 differentially expressed genes between FB and BR2.A total of 3,769 differentially expressed genes were found between BR2 and BR5 (Fig. 2B).Subsequently, these 37,842 differentially expressed genes were analyzed on the basis of clustering expression trends over time using STEM software.The results showed that these differentially expressed genes exhibited 50 different expression patterns within 7 periods.Among them, 20,709 differentially expressed genes showed 8 significantly clustered expression patterns (p < 0.05).The most critical of these patterns included expression pattern I, which showed downregulation, and expression pattern II, which showed downregulation followed by upregulation.In addition, differentially expressed genes in expression pattern 22 were upregulated from BR1 to BR2, downregulated from BR2 to BR3, and then upregulated from BR3 to BR4 and from BR5 to BR6, while expression pattern 8 showed a trend of downregulation followed by upregulation (Fig. 2C).BgIAA8 was downregulated and then upregulated, BgEIL1 was upregulated, and BgIAA9 showed a decreasing trend.The BgSCR gene, a leaf development-related gene, was also expressed in bracts (Fig. S2).
GO enrichment analysis revealed that these differentially expressed genes were involved in important pathways, such as reproduction, reproductive processes and growth (Fig. 2D).The SBP and MADS-box families were the main families involved.Among them, members of the MADS-box family include BgAP1, BgFULL, and BgCMB1, while members of the SBP family include SPL16, SPL8, and SPL14.(Fig. 2E, F, G).These results further revealed the important role of gene expression regulation in plant bract development.KEGG enrichment analysis revealed that the metabolic pathways related to bract development were mainly related to porphyrin and chlorophyll metabolism, plant hormone signal transduction and other metabolic pathways (Fig. 2H, Fig. S3).

Chlorophyll metabolic pathways during bract development
The color of B. glabra 'Mrs.Eva White' bracts changes from green to white during their development, a transition that is regulated by chlorophyll metabolism.Chlorophyll metabolism consists of three main processes: synthesis, recycling and degradation.In the synthetic pathway, hydroxymethyl chlorophyll is gradually reduced to chlorophyllide by the catalysis of NADPH-POR.BgPORA was highly expressed in the early stage of bract development, and its expression gradually decreased to very low levels as development progressed, suggesting that the ternary complex formed by hydroxymethyl chlorophyll a, NADPH, and BgPOR is the key to the green color of bracts in the early stage of bract development (Fig. 3).The genes found to interact with BgPORA by differential protein network analysis were BgPPOC, BgPAO, and BgCHLH (Fig. S4).The expression of the BgSGR, BgPPH, BgPAO and BgRCCR genes decreased at the early stage of bract development but then gradually increased, especially at the BR3 and BR4 stages.Therefore, it can be hypothesized that this period is the critical period for change in bracts color from green to white.

Metabolism of auxin and abscisic acid during bract development
Endogenous plant hormones play an important role in leaf development and regulate the development of B. glabra bracts.Further in-depth analysis of the plant hormone signaling pathways led to the identification of many differentially expressed genes involved in hormone signaling response and transduction at key sites of bract formation.These genes are associated with a variety of hormones, such as auxin, abscisic acid, cytokinin, gibberellins, ethylene, and jasmonic acid.
Indole-3-acetic acid (IAA) synthesis can be divided into the tryptophan-dependent pathway and the tryptophanindependent pathway.In the growth hormone signal transduction pathway, we further identified 44 homologous genes that encode 12 enzymes in the growth hormone synthesis pathway.The tryptophan-independent pathway includes 1 BgASB1 gene, 1 BgTRPX gene, five BgPAT1 genes, two BgPAI1 genes, and six BgTRPC genes.The BgTRPX, BgASB1, BgPAT1, BgPAI1, BgPRPC, and BgTRA2 genes in this pathway are highly expressed in the early stages of bract development and decrease in the later stages.The tryptophan-dependent pathway includes 15 BgTRPB genes, 4 BgTIR1 genes, 3 BgAMI1 genes, and 1 each of the BgALDO2, BgTAR1, BgYUC , and BgTRPA2 genes.The expression of the BgYUC and BgAMI1 genes of this pathway increased in the early stage.The expression of the BgYUC gene started to decrease gradually in the BR2 period, while that of the BgAMI1 gene started to decrease gradually in the BR3 period, and the expression of the BgALDO2 gene increased with bract development.These results suggest that these genes may play important regulatory roles in bract development (Fig. 4).
In the abscisic acid signaling pathway, we further identified 23 homologous genes encoding four enzymes involved in the growth hormone synthesis pathway.These genes included 2 BgECED2 genes, 3 BgECED1 genes, 5 BgALDO3 genes, and 12 BgABA2 genes.BgNCED2 was upregulated during BR5, and the BgNCED1 and BgALDO3 genes were upregulated during BR6.The BgABA2 gene was upregulated during early development.Therefore, it is inferred that these genes play important regulatory roles in bract senescence (Fig. 5).

Weighted gene correlation network analysis (WGCNA)
A systems biology approach using weighted gene correlation network analysis (WGCNA) was applied, using 12,370 genes (FPKM ≥ 10) and 1,506 transcription factors, as a way to reveal the function of the network.In this network, the blue and brown modules showed a significant negative correlation with bract area and chlorophyll content, while the variability was particularly significant for the red and brown modules (Fig. 6A, Fig. S5).
Several key HUB genes were identified, including Bg_3140, Bg_11315, and Bg_23959 in MEblue (Fig. 6B, Fig. S6); the transcription factor HUB genes Bg_26128 and Bg_10088 in MEbrown (Fig. 6C, Fig. S7); and structural genes with HUBs, mainly Bg_23913 and Bg_37656 (Fig. 6D).Notably, the HUB genes BgPORA and BgEXLA1 showed significant differences in expression during BR1.Among them, the HUB gene EXLA1 encodes an expansion protein that plays an important role in plant cell wall relaxation and regulates the expansion of B. glabra bract cells.The HUB gene PORA, on the other hand, is a key gene in the process of chlorophyll metabolism and participates in the biosynthesis of light-sensitive pigments by regulating the expression of PORA, which in turn affects chlorophyll biosynthesis, which is consistent with chlorophyll degradation during bract development in Trigonella foetidum (Fig. 6D).
In addition, we also found that the brown module HUB genes included BgBZP44, BgAPRR5 and BgIAA7, which  6E). BgTLP8, BgAPRR2, BgBZP44, BgEXLA1, and BgPORA, which play important roles in the regulation of B. glabra bract development and senescence and other processes, were highly expressed in the blue module during the early stages of bract development.

qRT-PCR of bract development-related candidate genes
Based on the above analysis, we performed qRT-PCR on three structural genes and nine transcription factors of the screened chlorophyll metabolic pathway.The qRT-PCR results were also compared with the RNA-seq (FPKM) results to verify the consistency of the gene expression pattern with the sequencing results.The results were similar to the expression trends obtained by RNA-Seq (Fig. 7).By correlation analysis, BgAP1 was found to be positively correlated with BgCMB1, BgDEFA, BgALDO2, BgALDO3, and BgSPL8, and BgAP1 was inversely correlated with BgNFYA3, BgPORA, BgAPRR5, BgSPL16, and BgFULL (Fig. S8).

Involvement of MADS-box proteins in B. glabra bract development
Flower development has been widely studied, but there are relatively few studies on bract development in ornamental plants, and the molecular mechanism underlying bract development is still unclear.MADS-box proteins are a large family of transcription factors that play key roles in eukaryotic development, regulating the intensity of target gene expression spatiotemporally by binding to cis-acting elements that interact with the target gene [15].In this study, we found that the genes BgAP1, BgCMB1, BgDEFA, and BgFULL, which were differentially expressed during the development of B. glabra bracts, were all in the MADS-box family and that the expression of BgAP1 was strongly inversely related to that of BgFULL.It has been suggested that FRUITFULL (FULL) is negatively regulated by AP1 during the early stages of Arabidopsis thaliana development.FULL is hardly expressed in early trophic organs until after the onset of inflorescence development [16].AP1 represses FULL expression during sepal and petal development.AP3, on the other hand, inhibits the expression of FULL during stamen development [17].Although it is unclear whether FULL is negatively regulated by AP1 during bract development, it is hypothesized that mutual regulation of BgAP1 and BgFULL may occur, based on their expression patterns in different developmental periods.Specifically, BgAP1 was expressed mainly in the later stages of bract development, whereas BgFULL was expressed in the early stages of bract development.This finding is more consistent with the results of previous studies.While most of the MADS-box genes are expressed only in flowers, FULL is expressed at different specific stages of bract development.Investigations in a variety of plant species suggest that the requirement for B-class gene function during heterotopic petaloidy varies dramatically, and these genes may function early, late, or at all during the development of petaloid organs [15,18].In Aristolochia (Aristolochiaceae, basal angiosperms), the expression of an AP3-like gene was detected only during the late stages of petaloid perianth development, and the expression of B-class paralogs was detected in the late development of Aquilegia petaloid sepals, suggesting that B-class homologs may only be required relatively late in the development of petaloid organs in these species [19].
CMB1 is a gene of the MADS-box family that is involved in the regulation of pistil and sepal development Fig. 7 Candidate gene qRT-PCR-identified FPKM values were derived from B. glabra transcriptome data.The actin gene was used as an internal control.LB as a control was assigned an arbitrary value of 1.0.The data represent three biological replicates and their mean values, and the error line represents the standard deviation of the three biological replicates in some plants.It has been suggested that PpCMB1 regulates pistil and sepal development in Prunus persica [20].CMB1 is involved in fruit ripening and sepal development [21].In Solanum lycopersicum, SlCMB1 is a SEPlike MADS-box gene that participates in inflorescence and sepal development via interactions with other genes [22].In this study, the expression of BgCMB1 tended to increase with bract development and was positively correlated with BgAP1 expression.BgAP1, BgFULL, BgCMB1, and BgDEFA were barely expressed in leaves but were highly expressed during bract development.These results suggest a potential role for BgCMB1 in bract development, possibly because BgCMB1 interacts with BgAP1 and BgFULL to regulate bract development.

The role of the SBP family in B. glabra bract development
SBPs are plant-specific class of transcription factors that are closely related to plant inflorescence development and yield [23]. SBPs have a zinc finger structure that recognizes and binds to the promoter of the MADS-box gene SQUAMOSA (SQUA), which is involved in plant growth and development as well as a variety of physiological and biochemical processes.In addition, SBPs, a class of transcription factors, can regulate flower and fruit development [24].In A. thaliana, the SBP homologs SPL3, SPL4 and SPL5 regulate A. thaliana flower development, whereas the SPL8 and SPL14 genes regulate pollen development [25], which in turn affects yield.
The MADS-box transcription factor FULL, together with a SBP transcription factor, plays a role in reproductive transformation and meristem identity transformation [26].AtSPL3 in A. thaliana was identified as a direct upstream activator of FULL, and the activation of FULL by SPL3 was very strong [27].In A. thaliana, Yamasaki also demonstrated that AtAP1 is downstream of the AtSPL3 gene [28].Further studies showed that after silencing and overexpressing the PpSPL16 gene in P. persica, the expression of the PpFULL and PpAP2a genes was downregulated and upregulated, respectively, which further regulated fruit ripening in P. persica [29].Whether SPL16 interacts with FULL during bract development is unknown, but the analyses in this study revealed that both BgSPL16 and BgAP1, as well as BgSPL16, play a crucial role in B. glabra bract development.In this study, the BgSPL genes exhibited different temporal and spatial expression patterns.The high expression levels of BgSPL8, BgSPL13 and BgSPL16 at the floral differentiation stage further confirmed the role of SPLs in regulating the asexual-to-reproductive transition through different regulatory roles during flower bud differentiation.SPL3 does not play a major role in asexual changes or anthesis, but it promotes a shift in floral phloem identity by activating floral phloem-specific genes [30].In addition, SPL7 and SPL8 induced phase transition and flowering in Gramineae by directly upregulating SEPALLATA3 (SEP3) and MADS32 [31].SPL8 was also found to be involved in GA signaling and positively regulated trichome formation on sepals and stamen filament elongation [32].In this study, we found that BgSPL8 was expressed in bracts but not in leaves during bract development, and BgSPL16 was highly expressed during the FB period, with lower expression observed during other periods.Therefore, in the present study, we hypothesized that BgSPL8 and SPL16 are associated with bract development, which provides important insight for further studies on plant growth and development.

Chlorophyll metabolism during bract development in B. glabra
In this study, B. glabra 'Mrs.Eva White' bract pigmentation was found to involve chlorophyll, carotenoids, and flavonoids, and at the beginning of the developmental stage, B. glabra bracts had a high chlorophyll content; however, as the bracts developed, the color gradually changed to white.Recently, phytochrome-mediated light signaling was reported to induce PORA gene expression in monocotyledons such as Hordeum vulgare and, similarly, in dicotyledons such as A. thaliana [33,34].Moreover, it has been suggested that PORA is abundant in yellowing plants, is active mainly during seedling deyellowing, and acts synergistically with the light-trapping complex [35].A mutant of A. thaliana with yellow leaves that did not turn green when grown in white light was identified, confirming that the presence of PORA confers a certain degree of photoprotection to the plant.The reaction of PORA with pchlide is the only step in which a light-requiring enzyme mediates the biosynthesis of chlorophyll in higher plants [36], and its expression is regulated by photosensitive pigments, thus affecting chlorophyll biosynthesis.This study further revealed that BgPORA may be a key gene for chlorophyll formation during bract development.
In addition, the STAY-GREEN protein (SGR) plays key regulatory roles in chlorophyll degradation and functions independently of PAO enzymes.Moreover, alterations in the transcription levels of genes encoding chlorophylldegrading enzymes, including PPH, PAO and RCCR , which have been identified in model crop species such as Oryza sativa and A. thaliana, affect the process of chlorophyll degradation and, consequently, the yellowing or senescence phenotypes of the leaves.It has been suggested that knockdown or inhibition of SGR expression delays chlorophyll degradation, leading to a stagnant green phenotype in plants during natural development or dark-induced senescence [37].In this study, B. glabra bracts were found to be upregulated during development with BgSGR in the later stages of B. glabra bract development, and BgSGR may be a key gene regulating the whitening of green bracts.

Effects of auxin and abscisic acid during bract development
Auxin is a phytohormone that plays an important role in plant growth and development.It has an unsaturated aromatic ring and an acetic acid side chain, in which indole-3-acetaldoxime can be converted to indole-3-acetaldehyde, and it can be catalytically converted to IAA by the enzyme ALDO2.This pathway is currently relatively understudied, especially with regard to bract development.The indole-3-pyruvic acid (IPA) pathway is the main pathway for IAA biosynthesis in plants and is common in most plant species [38].It has been shown that YUCCA (YUC ) is involved in the IPA pathway in synergy with tryptophan aminotransferase (TAA) of Arabidopsis, wherein TAA is involved in catalyzing the conversion of tryptophan to IPA, and subsequently, YUC is involved in catalyzing the production of IAA from IPA [39,40].The YUC and TAA families mainly regulate embryonic development after the spherical embryo stage [41].In this study, we found that the BgYUC gene is highly expressed mainly during the prophase of the growth hormone synthesis pathway and plays a regulatory role in the prophase of bract development.In this study, we hypothesized that BgYUC expression in this pathway is high in the early stages of bract development and low in the later stages, but the underlying molecular mechanism in bract development remains to be investigated.
The abscisic acid metabolic pathway mainly includes the terpenoid pathway and the carotenoid pathway [42].The terpenoid pathway, also known as the C15 direct pathway, consists of the direct formation of 15-carbon ABA from farnesyl pyrophosphate (FPP) via cyclization and oxidation, whereas the carotenoid pathway, also known as the C40 indirect pathway, is the main pathway for major BgABA synthesis in higher plants.In this pathway, the conversion of zeaxanthin to violaxanthin is catalyzed by zeaxanthin epoxidase (ZEP), which is followed by 9-cis-epoxy carotenoid dioxygenase (NCED)-mediated catalysis of the production of xanthoxin from 9-cis-neoxanthin.The shortchain alcohol dehydrogenase BgABA2 is converted to ABA-aldehyde, which is ultimately converted to ABA by ALDO3 [43].In this study, we found that BgALDO3 was highly expressed during the BR6 period at the end of bract development, suggesting that bract senescence is associated with BgALDO3.It has been suggested that the ABA signaling pathway is associated with pseudoresponse regulator 5 (APRR5) and pseudoresponse regulator 7 (APRR7) and that ABI5, a key regulator in the ABA signaling pathway, specifically interacts with APRR5 and APRR7 [44].In this study, BgAPRR5 was found to be highly expressed during the BR6 period, and it was hypothesized that this gene is associated with the abscisic acid metabolic pathway, which provides important clues for understanding the molecular mechanisms underlying the effects of plant auxin and abscisic acid in bract development.

Conclusion
In conclusion, our investigation into the development of B. glabra bracts revealed critical physiological and genetic mechanisms involved.We observed key changes in bract morphology, including increased area and chlorophyll content, accompanied by a reduction in hairiness (glabrescence).These morphological changes strongly correlate with the dynamics of hormone transduction pathways and chlorophyll metabolism.Importantly, genes within the MADS-box and SBP families, such as BgAP1, BgFULL, BgSPL16, BgCMB1, and BgDEFA, play pivotal roles in these developmental processes.Our study also highlights the significance of the BgPORA and BgSGR genes in the biosynthesis and breakdown of chlorophyll during bract development.The expression patterns of BgPRPC and BgTRA2 suggest their involvement in early developmental stages through both tryptophan-dependent and tryptophanindependent growth hormone pathways.Furthermore, the upregulation of BgALDO3 during the BR6 period underscores its potential importance in later developmental stages, particularly in the abscisic acid pathway.These findings not only enhance our understanding of bract development in B. glabra but also suggest avenues for future research to explore the regulatory mechanisms of floral architecture in ornamental plants.

Plant material and sample collection
Bougainvillea glabra 'Mrs.Eva White' planted at the flower base of Hunan Agricultural University was selected as the plant material for this study in August 2021, and the bract developmental stages were divided into eight stages for bract sample collection (Fig. 1A).Three plants were set up as 1 biological replicate, and 3 biological replicates were obtained at each developmental stage.Twenty representative samples were collected from each biological replicate and mixed.These samples were immediately placed in liquid nitrogen, stored at -80 °C for RNA extraction and then sent to Fraser Bioinformatics for sequencing using a PacBio sequencer.

Microscopic observation, paraffin sectioning, and scanning electron microscopy
Dissection of bracts at the flower bud stage was performed to observe their internal structure.Buds from both the LB and FB periods were selected for paraffin sectioning.Samples from the LB and FB periods were soaked in FAA fixative, and the subsequent experiments were performed by Wuhan Safeway Biotechnology Co.To understand flower bud development via resin sectioning, buds from different periods were collected, immediately soaked in FAA fixative (70% ethanol:glacial acetic acid:formaldehyde: 90:5:5) and dehydrated after embedding using a Technovit 7100 resin embedding kit.After embedding, the resin was sectioned with a Leica EM UC6 microtome, and 0.1% toluidine blue dye solution was used for staining for microscopy.To observe the phenotypic changes in bract development, samples were taken from the BR1, BR3, BR5, and LE periods for electron microscopy scanning, and the subsequent experiments were performed at Wuhan Sevier Biotechnology Co.

Bract area and chlorophyll measurement
B. glabra 'Mrs.Eva White' bracts exhibited an increase in area and faded from green to white during growth.The rate of bract development was observed daily, and the area of B. glabra 'Mrs.Eva White' bracts was measured using a leaf area meter.The chlorophyll and carotenoid contents of leaf buds, leaf blades and bracts at different periods of development were determined by ultraviolet-visible spectrophotometry.Several samples were harvested, washed and cut into strips of approximately 2 mm, weighed to 0.2 g, and extracted in 95% ethanol for 24 h, and three replicates were set up for each species in each treatment.After 24 h, the absorbance at 470 nm, 665 nm and 649 nm was measured by an ultraviolet-visible spectrophotometer, and the contents of chlorophyll a, chlorophyll b and carotenoids were calculated.

Iso-Seq analysis method
Total RNA was extracted from the tissue samples.The concentration and purity of the extracted RNA were tested by a Nanodrop 2000; the genomic contamination, purity and integrity of the RNA samples were assessed by agarose gel electrophoresis; and the RNA integrity number (RIN) values were determined by an Agilent 2100.High-quality RNA is the basis for successful sequencing.To ensure the accuracy of the sequencing data, the samples were tested to ensure that they met requirements before library construction.After library testing, fulllength transcriptome sequencing was performed using a PacBio sequencer according to the target downstream data volume.The raw sequencing data were preprocessed using SMRTlink software, and full-length transcript sequences were obtained via Iso-Seq analysis.

STEM and PCA
Gene expression levels were calculated as the number of exon model reads per kilobase per million mapped reads (FPKM).Based on the FPKM data of the differentially expressed genes at the seven bract developmental stages, the expression trends of the differentially expressed genes over time were analyzed by clustering using STEM software.PCA of the dataset was performed using the Omic-Share Tools tool (https:// www.omics hare.com/ tools/ home/ report/ repor tpca.html).

Identification of coexpression modules and visualization of hub genes
Based on the gene-level FPKM data of differentially expressed genes at the seven bract developmental stages, the R software package for WGCNA was used [45].The default minimum number of genes per module was 30, and the threshold for module merge correlation was 0.9 (Fig. S9).These modules were obtained using the automated network construction function block with default settings.Eigenvalues were calculated for each module and used to test for associations with area and chlorophyll content.The genes were grouped into 8 specific modules.The network was visualized using Cytoscape_v.3.8.0 [46].

Quantitative real-time PCR
The BgActin gene was used as the internal control.The primers used in the study are listed in Supplemental Table S1.Real-time fluorescence quantitative RT-PCR was performed using a ChamQ Universal SYBR qPCR Master Mix kit.The relative gene expression levels were normalized using the 2 −ΔΔCt method.

Data analysis
The experimental results are expressed as the mean ± standard error and were analyzed using Excel 2010 and SPSS 22.0.The significance of differences among different datasets was analyzed using Duncan's multiple range test at a significance level of p < 0.05.Heatmaps for this article were generated with OmicShare Tools (https:// www.omics hare.com/ tools/ home/ report/ repor tpca.html).Real-time fluorescence quantitative RT-PCR plots were generated with Origin software.

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See figure on next page.)Fig. 1 Morphostructural and chlorophyll content changes during bract development in B. glabra.A B. glabra leaf buds, bracts in 7 developmental periods and leaf sampling periods; LB (leaf bud), FB (floral bud), BR (bract), LE (leaf ).B Resin section of B. glabra during bud development.a Inflorescence primordium differentiation stage.b Late stage of floral primordial differentiation.c The organs of the third whorl begin to develop consecutively.C Electron microscope image of the bract development process of B. glabra.a Electron microscope image of bracts from the BR1 period.b Bract fluff shedding.c Electron microscopy image of bracts at BR5. D Changes in bract area during bract development in B. glabra.E Chlorophyll and carotene contents of B. glabra leaf buds, bracts and leaves at seven developmental stages

Fig. 2
Fig. 2 Analysis of genetic differences.A PCA plot of the sample FPKM values.B Venn diagrams showing differentially expressed genes among the LB/FB, FB/BR2, BR2/BR5, and BR5/LE comparisons.C STEM trend analysis plot.D KEGG enrichment analysis plot of differentially expressed transcription factors.E GO enrichment analysis plot of differentially expressed transcription factors.F Heatmap of significant expression trends of genes in the SPL family.G Heatmap of differentially expressed MADS-box family members with significant expression trends.H Differential protein interaction network map (See figure on next page.)